Preservative solution for heme protein, and method for stabilizing heme protein

ABSTRACT

An object of the present invention is to provide a novel preservative solution for a heme protein and a method for stabilizing a heme protein, which are effective against denaturation or degradation of a heme protein, and the present invention specifically relates to a preservative solution for a heme protein comprising a disulfonic acid or a salt thereof, and a method for stabilizing a heme protein, which involves bringing a disulfonic acid or a salt thereof into coexistence in a sample comprising a heme protein.

TECHNICAL FIELD

The present invention relates to a preservative solution for a hemeprotein and a method for stabilizing a heme protein. The presentinvention particularly relates to a preservative solution for a hemeprotein and a method for stabilizing a heme protein, which are usefulfor immunoassay methods.

BACKGROUND ART

In recent years, a fecal occult blood test for detection of fecal bloodhas been broadly carried out as a primary test for colon cancerscreening or a method for screening for a lower gastrointestinaldisease, with the increasing incidence of cancer. The fecal occult bloodtest is conducted by a chemical measurement method based on a chemicalcoloring reaction employing the peroxidase-like activity of hemoglobinthat is a heme protein or an immunoassay method employing a specificantibody against human hemoglobin. In particular the immunoassay methodenables convenient and rapid measurement without the need of any dietaryrestriction and any restriction of drug-taking before the test, comparedwith chemical measurement methods, and therefore it becomes establishedas a major testing method for the fecal occult blood test.

However, hemoglobin is known to be very unstable in a solution andeasily denatured or degraded. Such denaturation or degradation destroyshemoglobin conformation, resulting in decreased antigenicity. Therefore,the immunoassay method for measuring hemoglobin in such a case canproduce erroneous measurement results. Examples of reasons ofdenaturation or degradation of hemoglobin are varied and includeincreased storage temperatures, the passage of time, bacteria andenzymes. For example, it is known with regard to storage temperaturesthat hemoglobin in a solution is relatively stable in a frozen orrefrigerated state, but at room temperature or a temperature higher thanthe room temperature, denaturation or degradation of hemoglobinproceeds.

In particular, on a fecal occult blood test, feces are often collectedby subjects themselves at their places, and are provided for the test bysuspending the feces in a closed container containing a diluent forstool specimens. In such cases, fecal human hemoglobin may be left tostand in the solution for several days, or exposed to a high temperatureduring transportation via a transportation means such as a postalservice. Also, a hospital or a clinical laboratory sometime takes a longtime to have measurement results, since a large number of specimens andother test items are tested at such a facility. Under suchcircumstances, denaturation or degradation of hemoglobin tends to takeplace in a fecal occult blood test because of combined reasons such as atemperature rise and the passage of time.

Moreover, a fecal occult blood test often involves conductingmeasurement using an automatic analyzer capable of measuring manyspecimens accurately and rapidly. Upon measurement using an automaticanalyzer, periodic calibration and precision control are performed forthe automatic analyzer using a reference sample containing hemoglobin ata known concentration and a control sample containing hemoglobin at aknown concentration, since changes in reagents and the analyzer affectthe precision of the test results. Calibration involves measuring areference sample containing a plurality of measurement target substanceswith known concentrations by an automatic analyzer, preparing acalibration curve, and performing calibration for the automaticanalyzer. Precision control involves measuring a control samplecontaining measurement target substances with known concentrations by anautomatic analyzer, and determining the analytical precision dependingon if the measurement value is within a predetermined range. However, ifhemoglobin contained in such a reference sample and a control sample isdenatured or degraded, calibration and precision control cannot beperformed accurately, leading to erroneous measurement.

Therefore, various methods for stabilizing hemoglobin have been proposedto suppress denaturation or degradation of hemoglobin so as to produceaccurate measurement results. Examples of such methods that have beenproposed include: a method involving adding antibacterial agents such asthimerosal and chlorhexidine (e.g., see Patent Literature 1); a methodinvolving adding non-human animal hemoglobin (e.g., see PatentLiterature 2); a method involving adding the sera of non-human animals(e.g., see Patent Literature 3); a method involving adding anglycosidase type bacteriolytic enzyme (e.g., see Patent Literature 4); amethod involving adding sulfurous acid, disulfurous acid, or the like(e.g., see Patent Literature 5); a method involving adding acyl arginineesters and cationic surfactants (e.g., see Patent Literature 6); and amethod involving adding glyoxalic acid (e.g., see Patent Literature 7).

Then, the applicant of the present invention has already proposed amethod involving adding a water soluble transition metal complex such asa ferrocyan compound (e.g., see Patent Literature 8 and PatentLiterature 9), a method involving adding an enzymatic degradationproduct of hemoglobin (e.g., see Patent Literature 10), a methodinvolving adding transition metals (e.g., see Patent Literature 11), amethod involving adding organic acid such as malic acid (e.g., seePatent Literature 12), a method involving adding delipidated albumin(e.g., see Patent Literature 13), and a method involving addingiminocarboxylic acid (e.g., see Patent Literature 14), for example.

However, hemoglobin is very unstable, so that even these methods forstabilizing hemoglobin are problematic in preventing denaturation ordegradation of hemoglobin sufficiently.

CITATION LIST Patent Literature

-   Patent Literature 1: JP Patent Publication (Kokai) No.    63-271160A(1988)-   Patent Literature 2: JP Patent Publication (Kokai) No.    2-296149A(1990)-   Patent Literature 3: JP Patent Publication (Kokai) No.    4-145366A(1992)-   Patent Literature 4: JP Patent Publication (Kokoku) No.    5-69466B(1993)-   Patent Literature 5: JP Patent Publication (Kokai) No. 2000-258420A-   Patent Literature 6: JP Patent Publication (Kokai) No. 2009-222710A-   Patent Literature 7: JP Patent Publication (Kokai) No. 2013-257216A-   Patent Literature 8: JP Patent Publication (Kokai) No.    7-229902A(1995)-   Patent Literature 9: JP Patent Publication (Kokai) No.    11-118806A(1999)-   Patent Literature 10: JP Patent Publication (Kokai) No.    11-218533A(1999)-   Patent Literature 11: JP Patent Publication (Kokai) No. 2001-249132A-   Patent Literature 12: JP Patent Publication (Kokai) No. 2003-14768A-   Patent Literature 13: JP Patent Publication (Kokai) No. 2003-194825A-   Patent Literature 14: JP Patent Publication (Kokai) No. 2009-097956A

SUMMARY OF INVENTION Technical Problem

To solve such problems, an object of the present invention is to providea novel preservative solution for a heme protein and a method forstabilizing a heme protein, which are effective against denaturation ordegradation of a heme protein represented by hemoglobin.

Solution to Problem

The preservative solution for a heme protein of the present invention ischaracterized by containing disulfonic acid or a salt thereof.Furthermore, the method for stabilizing a heme protein of the presentinvention is characterized by bringing disulfonic acid or a salt thereofinto coexistence in a sample containing the heme protein.

Specifically, the present invention encompasses the following (1) to(10).

(1) A preservative solution for a heme protein, containing a disulfonicacid or a salt thereof.

(2) The preservative solution for a heme protein according to (1),wherein

the disulfonic acid or a salt thereof has at least one of an open chainhydrocarbon group and a cyclic hydrocarbon group, and

the disulfonic acid or a salt thereof is at least one selected from thegroup consisting of:

a disulfonic acid or a salt thereof in which the open chain hydrocarbongroup is a branched or linear hydrocarbon group and the main chain ofthe branched open chain hydrocarbon group or the linear open chainhydrocarbon group has any one of 1 to 10 carbon atoms;

a disulfonic acid or a salt thereof in which the cyclic hydrocarbongroup is a cycloalkylene group or an aryl group and the cyclichydrocarbon group has any one of 3 to 10 carbon atoms; and

a disulfonic acid or a salt thereof in which the cycloalkylene group orthe aryl group has one or more substituted nitrogen atoms.

(3) The preservative solution for a heme protein according to (1) or(2), wherein the disulfonic acid or a salt thereof is at least oneselected from the group consisting of methanedisulfonic acid,ethanedisulfonic acid, propanedisulfonic acid, butanedisulfonic acid,naphthalenedisulfonic acid, and piperazine-N,N′-bis(2-ethanesulfonicacid) or a salt thereof.

(4) The preservative solution for a heme protein according to any one of(1) to (3), wherein the concentration of the disulfonic acid or a saltthereof is 0.001 mol/L or more and 0.3 mol/L or less.

(5) The preservative solution for a heme protein according to any one of(1) to (4), further containingN-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid.

(6) The preservative solution for a heme protein according to any one of(1) to (5) further containing a heme protein, which is used as areference sample or a control sample.

(7) The preservative solution for a heme protein according to any one of(1) to (6), which is used for immunoassay.

(8) A method for stabilizing a heme protein, wherein a disulfonic acidor a salt thereof is brought into coexistence in a sample containing aheme protein.

(9) The method for stabilizing a heme protein according to (8), whereinthe concentration of the disulfonic acid or a salt thereof is 0.001mol/L or more and 0.3 mol/L or less.

(10) An immunoassay method for a heme protein, comprising a step ofbringing a heme protein into contact with an anti-heme protein antibodyin the presence of a disulfonic acid or a salt thereof.

This description includes part or all of the contents as disclosed inJapanese Patent Application No. 2015-070667 which is a priority documentof the present application.

Advantageous Effect of Invention

According to the preservative solution for a heme protein and the methodfor stabilizing a heme protein of the present invention, denaturation ordegradation of a heme protein can be suppressed and the heme protein canbe stably stored.

DESCRIPTION OF EMBODIMENTS

The present invention will be explained in detail hereinbelow.

The preservative solution for a heme protein of the present inventioncontains disulfonic acid or a salt thereof. Moreover, the method forstabilizing a heme protein of the present invention brings a disulfonicacid or a salt thereof into coexistence in a sample containing a hemeprotein.

Disulfonic acid or a salt thereof to be used in the present invention isnot particularly limited and can be selected from known examplesthereof. Disulfonic acid to be used in the present invention isdisulfonic acid having at least one open chain or cyclic hydrocarbongroup that may have a saturated or unsaturated bond, and in particular,the disulfonic acid is preferably disulfonic acid consisting of an openchain or cyclic hydrocarbon group and two sulfone groups. Disulfonicacid to be used in the present invention may have either an open chainhydrocarbon group or a cyclic hydrocarbon group, or both of thesegroups.

In particular, the open chain hydrocarbon group of disulfonic acid to beused in the present invention is a branched or linear hydrocarbon group.The main chain of the branched hydrocarbon group or the linearhydrocarbon group has any one of preferably 1 to 10 and more preferably1 to 4, and further preferably 1 or 2 carbon atoms. The main chain ofthe branched hydrocarbon group or the linear hydrocarbon group ispreferably an alkyl group, an alkenyl group, an alkynyl group, analkylene group, an alkenylene group, or an alkynylene group. Examples ofthe disulfonic acid having an alkylene group include methanedisulfonicacid, 1,2-ethanedisulfonic acid (hereinafter, referred to as 1,2-EDS),1,3-propanedisulfonic acid, 1,4-butanedisulfonic acid,1,5-pentanedisulfonic acid, and 1,6-hexanedisulfonic acid.

Furthermore, a cyclic hydrocarbon group of disulfonic acid to be used inthe present invention has preferably any one of 3 to 10 carbon atoms.The cyclic hydrocarbon group is preferably a cycloalkyl group, acycloalkenyl group, a cycloalkynyl group, or an aryl group. Inparticular, the aryl group is preferably a phenylene group or anaphthylene group. Examples of disulfonic acid having a phenylene groupor a naphthylene group include 1,2-benzenedisulfonic acid,1,3-benzenedisulfonic acid, 1,4-benzenedisulfonic acid,1,6-naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid, and2,7-naphthalenedisulfonic acid.

Furthermore, the cyclic hydrocarbon group may be a branched hydrocarbongroup. Also, the cyclic hydrocarbon group may be substituted with 1 ormore, preferably 1 to 3, or more preferably 1 to 2 nitrogen atoms. Anexample of disulfonic acid having a hydrocarbon group substituted with anitrogen atom(s) is piperazine-N,N′-bis(2-ethanesulfonic acid).

Furthermore, more preferably, disulfonic acid to be used in the presentinvention is disulfonic acid in which two sulfone groups are bound to anopen chain or cyclic hydrocarbon group. Moreover, disulfonic acid havingan open chain hydrocarbon group has two sulfone groups preferably atdifferent carbon atoms of the main chain of a branched hydrocarbon groupor a linear hydrocarbon group, and more preferably at each of theseends. Disulfonic acid to be used in the present invention is preferably1,2-ethanedisulfonic acid (1,2-EDS).

A hydrocarbon group of disulfonic acid to be used in the presentinvention may have a substituent such as a halogen group and/or ahydroxy group. Also, in disulfonic acid having a branched hydrocarbongroup, the branched chain preferably comprises hydrocarbon. Moreover,disulfonic acid or a salt thereof to be used in the present invention isat least one or may be a mixture of two or more of the disulfonic acidor a salt thereof.

According to the present invention, a preservative solution or a samplecontains a heme protein and disulfonic acid or a salt thereof, so thatdenaturation or degradation of the heme protein can be suppressed. Inparticular, according to the present invention, disulfonic acid containsethanedisulfonic acid, so that the stability of the heme protein can beenhanced more significantly. Disulfonic acid to be used in the presentinvention does not adversely affect measurement, and is particularlysuitable for immunoassay methods using latex immunoagglutination assay.

A salt of disulfonic acid to be used in the present invention is notparticularly limited, and is a monovalent, a divalent, or a trivalentmetal salt of disulfonic acid. Examples of such a salt of disulfonicacid include an alkali metal salt, an ammonium salt, an alkaline-earthmetal salt, a ferrate, and an aluminum salt. Examples of an alkali metalinclude lithium, sodium, potassium, rubidium, and cesium. Examples of analkaline-earth metal include calcium, strontium, barium, and radium.

The upper limit of the concentration of disulfonic acid or a saltthereof contained in the preservative solution for a heme protein or thesample containing a heme protein of the present invention is 0.3 mol/Lor less, more preferably 0.2 mol/L or less, and further preferably 0.15mol/L or less, and the lower limit of the same is 0.001 mol/L or more,more preferably 0.005 mol/L or more, further preferably 0.01 mol/L ormore, and most preferably 0.02 mol/L or more. If the concentration ofdisulfonic acid or a salt thereof is less than 0.001 mol/L, the effectof stabilizing the heme protein will be insufficient. On the other hand,the concentration of disulfonic acid or a salt thereof higher than 0.3mol/L will inhibit immunoreaction to affect the measurement more easily,as well as not provide the sufficient effect of stabilizing the hemeprotein.

A heme protein as target of the present invention and a heme proteincontained in a sample of the present invention can be adequatelyselected from proteins containing heme as a component. Examples of hemeproteins include hemoglobin, myoglobin, peroxidase and catalase. Inparticular, a heme protein as target of the present invention and a hemeprotein contained in a sample of the present invention is preferably aheme protein that is an immunological analyte, and is more preferablyhuman hemoglobin. In immunoassay methods, maintaining the antigenicityof a detection target is important. However, the antigenicity of a hemeprotein can be maintained according to the present invention, enablingmore accurate measurement of the heme protein. In particular, a hemeprotein as target of the present invention and a heme protein containedin a sample of the present invention may be hemoglobins in a biologicalsample, so that prevention of erroneous measurement results in diagnosisof diseases such as large bowel cancer can be expected. Examples ofhemoglobin include fecal hemoglobin, hemoglobin commercially availableas a reference sample or a control containing hemoglobin prepared fromerythrocytes, and lyophilized hemoglobin.

Moreover, the preservative solution for a heme protein or the samplecontaining a heme protein of the present invention can contain asolution in which a heme protein can be dissolved. Such a solution maybe a solution in which a heme protein can be dissolved and examplesthereof include buffer solutions. A buffer to be used for preparation ofa buffer solution is not particularly limited, as long as it has buffercapacity, and examples thereof include Good's buffer, phosphate buffer,Tris buffer, glycine buffer, and borate buffer.

Furthermore, examples of Good's buffer include2-morpholinoethanesulfonic acid (MES) buffer,bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer,N-(2-acetamide)iminodiacetate (ADA) buffer,piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer,N-(2-acetamide)-2-aminoethanesulfonic acid (ACES) buffer,3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer,3-morpholinopropanesulfonic acid (MOPS) buffer,N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) buffer,N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer,3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO)buffer, 2-hydroxy-3-{[N-tris(hydroxymethyl)methyl]amino}propanesulfonicacid (TAPSO) buffer, piperazine-N,N′-bis(2-hydroxypropane-3-sulfonicacid) (POPSO) buffer,N-(2-hydroxyethyl)-N′-(2-hydroxy-3-sulfopropyl)piperazine (HEPPSO)buffer, N-(2-hydroxy ethyl)-N′-(3-sulfopropyl)piperazine (EPPS) buffer,tricine[N-tris(hydroxymethyl)methylglycine] buffer,bicin[N,N-bis(2-hydroxyethyl)glycine] buffer,3[N-tris(hydroxymethyl)methyl]aminopropanesulfonic acid (TAPS) buffer,2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer,3-(N-cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) buffer, and3-(N-cyclohexylamino)propanesulfonic acid (CAPS) buffer. Particularly,in the present invention, among examples of Good's buffer, HEPES ispreferably used, the stability of a heme protein can be enhancedsignificantly by bringing a disulfonic acid or a salt thereof intocoexistence with the heme protein.

The buffer concentration is not particularly limited, as long as it isappropriate for measurement, and ranges from 0.001 to 2.0 mol/L,preferably 0.005 to 1.5 mol/L, and further preferably 0.01 to 1.0 mol/L.

Furthermore, the pH of the preservative solution for a heme protein orthe sample containing a heme protein of the present invention ispreferably within a neutral range, preferably ranges from 5 to 10, andis more preferably within a range of 6 to 8. The pH of less than 5, orthe pH of higher than 10 results in deteriorated stability of the hemeprotein, so that the heme protein will be easily denatured or degraded.The pH can be adjusted by known methods and may also be adjusted usingNaOH or an appropriate buffer.

Moreover, the preservative solution for a heme protein or the samplecontaining a heme protein of the present invention can contain a knownprotein protection agent, such as a water soluble transition metalcomplex, a ferrocyan compound, an enzymatic degradation product ofhemoglobin, transition metals, organic acid, iminocarboxylic acid,inactive proteins represented by albumin and gelatin, and sodium azide.The solution or the sample may also contain an antimicrobial agent, forexample, for preventing unnecessary microbial proliferation. Thesolution or the sample may further contain a salt, an aggregationaccelerating agent, and other components, if necessary, unless theadvantageous effects of the invention are not disturbed. According tothe present invention, the stability of a heme protein can be enhancedby the present invention in cooperation with a conventional proteinprotection agent, an antimicrobial agent, or the like without inhibitingthe action of the conventional protein protection agent, theantimicrobial agent, or the like.

Furthermore, when the preservative solution for a heme protein or thesample containing a heme protein of the present invention containsalbumin, bovine albumin, equine albumin, porcine albumin, sheep albumin,rabbit albumin, human albumin, rat albumin or the like can be used, anda serum containing albumin can also be used. The concentration ofalbumin in the preservative solution for a heme protein or the samplecontaining a heme protein of the present invention ranges from 0.0005 to2.0 w/v %, and more preferably ranges from 0.01 to 0.5 w/v %.

A measurement method for a heme protein is not particularly limited, andis an immunoassay method using an anti-heme protein antibody (antibodyspecifically binding to a heme protein), and is preferably animmunoassay method using an anti-human hemoglobin antibody.Specifically, such a method involves, in a sample, bringing a hemeprotein (for example, human hemoglobin) into contact with an anti-hemeprotein antibody (for example, anti-human hemoglobin antibody) in thepresence of disulfonic acid or a salt thereof, causing anantigen-antibody reaction, and detecting or measuring a heme protein inthe sample based on the resulting immunocomplex. When a heme protein ishuman hemoglobin, examples of immunoassay methods for human hemoglobininclude: single radial immunodiffusion that involves confirming theexpression of a precipitin line by an immunocomplex formed by binding ofan anti-human hemoglobin antibody and human hemoglobin in a test samplein an agar plate; a latex immunoagglutination assay using latexparticles sensitized with an anti-human hemoglobin antibody; an enzymeimmunoassay or a radioimmunoassay using an anti-human hemoglobinantibody labeled with an enzyme or a radioactive element; a gold colloidagglutination colorimetric method using gold colloid particlessensitized with an anti-human hemoglobin antibody; and animmunochromatography using an anti-human hemoglobin antibody labeledwith a metal colloid or the like and a capture antibody for capturing animmunocomplex of the anti-human hemoglobin antibody and human hemoglobinin a membrane such as a nitrocellulose membrane. Specifically, the lateximmunoagglutination assay involves reacting latex particles sensitizedwith an anti-human hemoglobin antibody with human hemoglobin in asample, and measuring human hemoglobin based on changes in turbidityresulting from latex agglutination due to the formation of animmunocomplex. Moreover, immunochromatography involves, in a membranesuch as a nitrocellulose membrane, supplying a sample, reacting humanhemoglobin in the sample with an anti-human hemoglobin antibody at alabeling reagent-retaining part for retaining the anti-human hemoglobinantibody labeled with a metal colloid or the like, so as to form animmunocomplex, causing the immunocomplex to move within the membrane bycapillary phenomenon, causing the immunocomplex to be captured by acapture antibody immobilized at a predetermined position of themembrane, and detecting human hemoglobin based on coloration resultingfrom the capture. Through the use of the preservative solution for aheme protein or the sample containing a heme protein of the presentinvention containing disulfonic acid or a salt thereof, any of thesemeasurement methods can protect the antigenic activity of a heme proteinand suppress errors in the measurement results.

The preservative solution for a heme protein of the present inventioncan be used as a solution for storing a heme protein in variousapplications. For example, the solution can be used as a solution fordissolving a heme protein derived from a biological sample such asfeces, urine, and blood, or a solution such as a diluent and an extractsolution. In particular, the solution is useful as a diluent for stoolspecimens in a test for detection of a heme protein, such as a fecaloccult blood test.

Furthermore, the preservative solution for a heme protein of the presentinvention may contain a heme protein as target of the above presentinvention, and can be used as various solutions containing hemeproteins. Similarly, the method for stabilizing a heme protein of thepresent invention can be applied to various samples containing hemeproteins. For example, the preservative solution for a heme protein andthe sample containing a heme protein of the present invention can beused as a reference sample containing a heme protein, a control samplecontaining a heme protein, and the like, and particularly as a referencesample containing a heme protein or a control sample containing the sameto be used for calibration or precision control for an automaticanalyzer. Even when a reference sample and a control sample containingheme proteins are stored for a long time period, the resultingmeasurement values of the heme proteins are required to remainunchanged. According to the present invention, denaturation ordegradation of heme proteins in a reference sample and a control samplecan be suppressed, even when the samples are stored at a relatively hightemperature, so that the invention can contribute to the stabilizationof the measurement values of heme proteins. Therefore, the preservativesolution for a heme protein and the sample containing a heme protein ofthe present invention are suitable as a reference sample and a controlsample containing heme proteins.

Furthermore, the preservative solution for a heme protein of the presentinvention can be provided as a kit for immunoassay of heme proteins(e.g., human hemoglobin), which is used for a fecal occult blood test,for example. This kit can contain, in addition to the preservativesolution for a heme protein of the present invention, a sample storagecontainer such as a feces collection container and an instruction manualfor the kit, and, when an immunoassay method is a lateximmunoagglutination assay, a latex solution sensitized with an anti-hemeprotein antibody, a diluent, and the like, or when an immunoassay methodis immunochromatography, an immunochromatography device (e.g., membrane,such as nitrocellulose membrane supported by a support having asample-supplying part, a labeling reagent-retaining part for retainingan anti-heme protein antibody labeled with a metal colloid or the like,and a detection part containing a capture antibody immobilized at apredetermined position), and the like.

The present invention will be further described specifically byreferring to examples, but the present invention is not limited by theseexamples.

EXAMPLES Example 1

A solution (pH7.0) containing 0.3 w/v % bovine serum albumin, NaOH, 0.05mol/L phosphate buffer solution as a buffer solution, and pure water asthe remainder was prepared. 1,2-EDS as an additive was added, to thesolution in such an amount as to give each concentration (0.01 to 0.2mol/L) listed in Table 1, thereby preparing a solution at eachconcentration. Hemolytic hemoglobin was added to 10 mL of the preparedsolutions in such an amount as to give a 600 ng/mL sample.

Immediately after addition of hemoglobin, the hemoglobin concentration(concentration immediately after addition) of each sample was measured.Subsequently, each sample was stored at 37° C. With the time of addinghemoglobin designated as 0 hour of storage, hemoglobin concentration ofeach sample was measured after 6 hours of storage and after 24 hours ofstorage (concentration after 6 hours of storage and concentration after24 hours of storage).

Hemoglobin concentration was measured using an OC sensor DIANA analyzer(Eiken Chemical Co., Ltd) and a dedicated reagent (OC-Hemodia Auto III:Eiken Chemical Co., Ltd) based on the measurement principle, a latexagglutination reaction; that is, a type of immunoassay method.Specifically, 35 μL of the sample was collected and used as a testsolution, and then 60 μL of latex (latex solution sensitized with 20 vol% anti-human hemoglobin rabbit polyclonal antibody) and 300 μL ofdiluent (11.92 mg/mL HEPES) were added to the test solution, followed bymeasurement of absorbance at a wavelength of 660 nm. Based on apreviously produced calibration curve, hemoglobin concentration in thetest solution was determined from the thus obtained measurement value.Measurement was performed in triplicate for each sample, the mean valueof the measurement results was employed as the hemoglobin concentrationof each sample.

From the thus measured hemoglobin concentrations, the residualpercentages of hemoglobin were found based on the following formula.

Residual percentage [%] of hemoglobin after 6 hours of storage or after24 hours of storage=100×hemoglobin concentration [ng/mL] after 6 hoursof storage or after 24 hours of storage/concentration [ng/mL]immediately after addition in control sample.

Specifically, the residual percentage of hemoglobin of each sample is arelative value with the hemoglobin concentration (immediately afteraddition) in a control sample designated as 100%. The control sample inthis example was a phosphate buffer solution (containing no 1,2-EDS)containing bovine serum albumin and NaOH, and the concentrationimmediately after addition in the control sample was 583 ng/mL. Theresults are shown in Table 1.

TABLE 1 Additive Residual percentage [%] Concentration After 6 hoursAfter 24 hours Buffer solution Additive [mM] of storage of storagePhosphoric acid (control) None 0 49.4 7.1 Phosphoric acid 1,2-EDS 1052.5 7.5 Phosphoric acid 1,2-EDS 20 56.3 9.0 Phosphoric acid 1,2-EDS 4061.9 12.8 Phosphoric acid 1,2-EDS 60 59.7 12.3 Phosphoric acid 1,2-EDS100 65.4 16.6 Phosphoric acid 1,2-EDS 150 71.5 24.3 Phosphoric acid1,2-EDS 200 46.2 7.9 (Note) Residual percentage is a relative value withthe concentration immediately after addition (583 ng/mL) in a controlsample designated as 100%.

As shown in Table 1, samples containing the disulfonic acid, 1,2-EDS,exhibited residual percentages after 6 hours of storage and after 24hours of storage higher than those of the control sample, indicatingthat 1,2-EDS had the effect of stabilizing hemoglobin. Moreover, theresidual percentages were found to be higher with the increasingconcentration of 1,2-EDS added, indicating that the increasing 1,2-EDSconcentration enhanced the effect of stabilizing hemoglobin.

Example 2

Samples were prepared in a manner similar to Example 1 except that 0.05mol/L N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (hereinafter,referred to as HEPES) was used instead of a phosphate buffer solutionand that 1,2-EDS was added in such an amount as to give eachconcentration (0.005 to 0.2 mol/L) listed in Table 2, and thenhemoglobin concentrations were measured. The results are shown in Table2. In addition, the residual percentage of each sample was representedby a relative value with the concentration immediately after addition(576 ng/mL) in a control sample (HEPES buffer solution (containing no1,2-EDS) containing bovine serum albumin and NaOH) designated as 100%.

TABLE 2 Additive Residual percentage [%] Concentration After 6 hoursAfter 24 hours Buffer solution Additive [mM] of storage of storage HEPES(control) None 0 61.8 11.9 HEPES 1,2-EDS 5 64.3 14.0 HEPES 1,2-EDS 1065.2 16.6 HEPES 1,2-EDS 20 67.1 18.1 HEPES 1,2-EDS 40 68.5 19.7 HEPES1,2-EDS 60 71.3 20.8 HEPES 1,2-EDS 100 72.4 21.6 HEPES 1,2-EDS 150 72.820.4 HEPES 1,2-EDS 200 73.1 21.8 (Note) Residual percentage is arelative value with the concentration immediately after addition (576ng/mL) in a control sample designated as 100%.

As shown in Table 2, samples containing the disulfonic acid, 1,2-EDS,exhibited residual percentages after 6 hours of storage and after 24hours of storage higher than those of the control sample, indicatingthat 1,2-EDS had the effect of stabilizing hemoglobin. In particular,because of containing HEPES and 1,2-EDS, even a sample having theconcentration of 1,2-EDS added of as low as 5 mM was found to have highresidual percentage after 6 hours of storage and after 24 hours ofstorage. Moreover, a higher residual percentages was found with theincreasing concentration of 1,2-EDS added, indicating that theincreasing 1,2-EDS concentration enhanced the effect of stabilizinghemoglobin.

Example 3

Samples were prepared in a manner similar to Example 1 except that 0.05mol/L HEPES, 0.05 mol/L piperazine-N,N′-bis(2-ethanesulfonic acid)(hereinafter, referred to as PIPES) or 0.05 mol/L2-morpholinoethanesulfonic acid (hereinafter, referred to as MES) wasused instead of a phosphate buffer solution, and that 1,2-EDS was addedin such an amount as to give each concentration listed in Table 3, andthen hemoglobin concentrations were measured. The results are shown inTable 3. In addition, the residual percentage of each sample wasrepresented by a relative value with the concentration immediately afteraddition (583 ng/mL) in a control sample (phosphate buffer solution(containing no 1,2-EDS) containing bovine serum albumin and NaOH)designated as 100%.

TABLE 3 Additive Residual percentage [%] Concentration After 6 hoursAfter 24 hours Buffer solution Additive [mM] of storage of storagePhosphoric acid (control) None 0 49.4 7.1 Phosphoric acid 1,2-EDS 2056.3 9.0 HEPES None 0 46.3 9.3 HEPES 1,2-EDS 20 67.6 20.2 PIPES None 050.4 9.2 PIPES 1,2-EDS 20 63.6 14.3 MES None 0 54.6 11.7 MES 1,2-EDS 2065.9 18.7 (Note) Residual percentage is a relative value with theconcentration immediately after addition (583 ng/mL) in a control sampledesignated as 100%.

As shown in Table 3, the effect of stabilizing hemoglobin exhibited by1,2-EDS was confirmed also in samples containing different buffersolutions. In particular, samples containing HEPES and 1,2-EDS exhibitedresidual percentages after 6 hours of storage and after 24 hours ofstorage significantly higher than those of a sample containing HEPES asa buffer solution but containing no 1,2-EDS, indicating that the samplescontaining HEPES and 1,2-EDS had extremely high effect of suppressingdenaturation or degradation of hemoglobin, as well as suggesting thatthe same can stabilize hemoglobin in the long term. Further, a samplecontaining HEPES and 1,2-EDS exhibited residual percentages after 6hours of storage and after 24 hours of storage higher than those of asample containing Good's buffer (PIPES or MES) and 1,2-EDS, and wasfound to have more enhanced effect of suppressing denaturation ordegradation of hemoglobin, indicating that it has a synergistic effectof stabilizing hemoglobin.

Example 4

Samples were prepared in a manner similar to Example 1 except that1,4-butanedisulfonic acid (hereinafter, referred to as 1,4-BDS),2,6-naphthalenedisulfonic acid (hereinafter, referred to as 2,6-NDS) orPIPES, instead of 1,2-EDS, was added in such an amount as to give eachconcentration listed in Table 4, and that 0.05 mol/L HEPES was usedinstead of a phosphate buffer solution, and then hemoglobinconcentrations were measured. The results are shown in Table 4. Inaddition, the residual percentage of each sample was represented by arelative value with the concentration immediately after addition (548ng/mL) in a control sample (phosphate buffer solution (containing nodisulfonic acid) containing bovine serum albumin and NaOH) designated as100%.

TABLE 4 Additive Residual percentage [%] Concentration After 6 hoursAfter 24 hours Buffer solution Additive [mM] of storage of storagePhosphoric acid (control) None 0 49.8 6.2 Phosphoric acid 1,4-BDS 5068.3 14.5 Phosphoric acid 1,4-BDS 150 69.9 18.4 Phosphoric acid 2,6-NDS50 55.7 11.1 Phosphoric acid PIPES 50 58.4 10.4 HEPES None 0 64.9 12.5HEPES 1,4-BDS 150 74.7 28.4 (Note) Residual percentage is a relativevalue with the concentration immediately after addition (548 ng/mL) in acontrol sample designated as 100%.

As shown in Table 4, the disulfonic acid, 1,4-BDS, 2,6-NDS, and PIPESwere found to have the effect of stabilizing hemoglobin.

Comparative Example 1

Samples were prepared in a manner similar to Example 1 except that8-anilino-1-naphthalenesulfonate (hereinafter, referred to as ANS) orsodium 2-mercaptoethanesulfonate (hereinafter, referred to as MESS) wasadded in such an amount as to give 0.01 mol/L instead of 1,2-EDS, andthen hemoglobin concentrations were measured. The results are shown inTable 5. In addition, the residual percentage of each sample wasrepresented by a relative value with the concentration immediately afteraddition (583 ng/mL) in a control sample (phosphate buffer solution(containing no additive) containing bovine serum albumin and NaOH)designated as 100%.

TABLE 5 Additive Residual percentage [%] Concentration After 6 hoursAfter 24 hours Buffer solution Additive [mM] of storage of storagePhosphoric acid (control) None 0 49.4 7.1 Phosphoric acid 1,2-EDS 1052.5 7.5 Phosphoric acid ANS 10 2.6 2.5 Phosphoric acid MESS 10 33.4 3.3(Note) Residual percentage is a relative value with the concentrationimmediately after addition (583 ng/mL) in a control sample designated as100%.

As shown in Table 5, denaturation or degradation of hemoglobin mighthave proceeded in a sample containing not disulfonic acid but sulfonicacid.

Therefore, it was demonstrated that a preservative solution and a samplecontaining disulfonic acid such as 1,4-BDS, 2,6-NDS, and 1,2-EDS canmaintain the residual percentages of hemoglobin after 6 hours of storageand after 24 hours of storage at high levels, even when stored at a hightemperature of 37° C. These results indicate that the preservativesolution and the sample containing disulfonic acid of the presentinvention suppress denaturation or degradation of hemoglobin and havethe effect of stabilizing hemoglobin, even subjected to temperature riseand the passage of time.

Moreover, when a stool specimen was added to each sample of Examples 1to 4 and Comparative example 1 and similar measurements were performed,tendency similar to that of measurement results in Examples 1 to 4 andComparative example 1 was observed.

It was revealed that according to the preservative solution for a hemeprotein containing disulfonic acid or a salt thereof and the method forstabilizing a heme protein of the present invention, denaturation ordegradation of a heme protein can be suppressed and the heme protein canbe stabilized.

INDUSTRIAL APPLICABILITY

According to the preservative solution for a heme protein and the methodfor stabilizing a heme protein of the present invention, denaturation ordegradation of a heme protein can be suppressed, and the heme proteincan be stably stored. Specifically, heme proteins such as those in adiluent for stool specimens in a fecal occult blood test, a referencesample containing a heme protein, and a control sample containing a hemeprotein can be stably stored.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1-10. (canceled)
 11. A preservative solution for a heme protein,comprising a disulfonic acid or a salt thereof, wherein the disulfonicacid or a salt thereof has at least one of an open chain hydrocarbongroup or a cyclic hydrocarbon group, and the disulfonic acid or a saltthereof is at least one selected from the group consisting of: adisulfonic acid or a salt thereof in which the open chain hydrocarbongroup is a branched or linear hydrocarbon group, does not have a cyclichydrocarbon group, and the main chain of the branched open chainhydrocarbon group or the linear open chain hydrocarbon group has any oneof 1 to 10 carbon atoms; and a disulfonic acid or a salt thereofselected from the group consisting of 1,2-benzenedisulfonic acid,1,3-benzenedisulfonic acid, 1,4-benzenedisulfonic acid and a disulfonicacid having a naphthylene group, or a salt thereof.
 12. The preservativesolution of claim 11, wherein the disulfonic acid consists of an openchain or cyclic hydrocarbon group and two sulfone groups.
 13. Thepreservative solution of claim 11, wherein the disulfonic acid has asubstituent and the substituent is a halogen group and/or a hydroxygroup.
 14. The preservative solution of claim 11, wherein the disulfonicacid or a salt thereof is at least one selected from the groupconsisting of methanedisulfonic acid, ethanedisulfonic acid,propanedisulfonic acid, butanedisulfonic acid, naphthalenedisulfonicacid, and a salt thereof.
 15. The preservative solution of claim 11,wherein the concentration of the disulfonic acid or a salt thereof is0.001 mol/L or more and 0.3 mol/L or less.
 16. The preservative solutionof claim 11, further comprisingN-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid.
 17. Thepreservative solution of claim 11, further comprising a heme proteinused as a reference sample or a control sample.
 18. The preservativesolution of claim 11, which is used for immunoassay.